Chemistry harbors the potential to provide ready access to natural proteins as well as to create nonnatural ones with desirable attributes. In the previous funding period, we used a variety of chemical and biochemical tools to reveal new structure-function relationships in one particular protein, ribonuclease A. During the course of that work, we discovered that two peptides, one with a C-terminal phosophinothioester and the other with an N-terminal azide, can be coupled by the "Staudinger ligation". This reaction proceeds rapidly in high yield, and is traceless - no residual atoms remain in the product. A major focus of the current proposal is to develop the Staudinger ligation as a tool for modem protein chemistry. The Specific Aims of this proposal are (1) to analyze the kinetics and mechanism of the Staudinger ligation of peptides by varying the amino acid residues at the junction, the phosphine, and the reaction conditions, and using time-resolved NMR spectroscopy to monitor the ligation reactions, (2) to develop a water-soluble phosphinothiol for the Staudinger ligation of proteins and peptides, (3) to develop an immobilized phosphinothiol that allows for Staudinger ligation to a soluble azide concomitant with release from the solid support, (4) to assemble two model proteins by Staudinger ligation of their component peptides in a process that is amenable to automation, (5) to evaluate the ability of reverse-turn mimics and a covalent mimic of a hydrogen bond to enhance the conformational stability of a protein, and (6) to determine the three-dimensional structure of synthetic and semisynthetic proteins described in this proposal, so as to enhance the interpretation of data on their conformational stability, folding, and function. Significance. The proposed research would provide new insight into the fundamental chemistry of proteins, as well as extend the capacity to access and manipulate proteins. We anticipate that this knowledge would have a high impact on biomedicine in this post-genomic era.